Crystal structure of SARS-CoV-2 main protease in complex with protease inhibitor PF-07321332

experiment;


Cloning, protein expression, and purification of SARS-CoV-2 M pro
The cell cultures were grown and the protein was expressed according to a previous report (Jin et al., 2020). The cell pellets were resuspended in lysis buffer (20mM Tris-HCl pH 8.0, 150 mM NaCl, 5% Glycerol), lysed by high-pressure homogenization, and then centrifuged at 25,000g for 30 min. The supernatant was loaded onto Ni-NTA affinity column (Qiagen, Germany), and washed by the lysis buffer containing 20 mM imidazole. The His-tagged M pro was eluted by lysis buffer containing 300 mM imidazole. The imidazole was then removed through desalting. Human rhinovirus 3C protease was added to remove the C-terminal His tag. SARS-CoV-2 M pro was further purified by ion exchange chromatography. The purified M pro was transferred to 10 mM Tris-HCl pH 8.0 through desalting and stored at -80 degrees until needed.

Crystallization, data collection, and structure determination
PF-07321332 is synthesized by Prof. Ma's Lab. SARS-CoV-2 M pro (6 mg/ml) was incubated with 1 mM PF-07321332 for 1 hour at room temperature and the complex was crystallized by hanging drop vapor diffusion method at 20 °C. The best crystals were grown using a well buffer containing 0.1 M MES pH 6.0, 5% polyethylene glycol (PEG) 6000, and 3% DMSO. The cryo-protectant solution was the reservoir but with 20% glycerol added.
X-ray data were collected on beamline BL19U1 at Shanghai Synchrotron Radiation Facility (SSRF) at 100 K and at a wavelength of 0.97852 Å using a Pilatus3 6M image plate detector. Data integration and scaling were performed using the program XDS (Kabsch, 2010). The structure was determined by molecular replacement (MR) with the PHASER (McCoy et al., 2007) and Phenix 1.19.2 (Liebschner et al., 2019) using the SARS-CoV-2 M pro (PDB accession number: 6LU7) as a search template. The model from MR was subsequently subjected to iterative cycles of manual model adjustment with Coot 0.8 (Emsley et al., 2010) and refinement was completed with Phenix REFINE (Afonine et al., 2012). The inhibitor PF-07321332 was built according to the omit map. The phasing and refinement statistics are summarized in Table S1. Coordinates and structure factors have been deposited in PDB with the accession number 7VH8.

Intact protein analysis
1 μL PF-07321332 (10mM in DMSO) was add into 50uL of the proteins (1 mg/mL). The mixtures were kept in room temperature for 30 min. Liquid chromatography-mass spectrometry (LC-MS) analyses were performed in positive-ion mode with an Agilent 6550 quadrupole-time-of-flight (QTOF) mass spectrometer (Santa Clara, CA) coupled with an Agilent 1260 high-performance liquid chromatograph (HPLC; Santa Clara, CA) for detecting the molecular weight of intact proteins. The samples were eluted from a Phenomenex Jupiter C4 300Å LC Column (2×150 mm, 5 μm) over a 15 min gradient from 5% to 100% acetonitrile containing 0.1% formic acid at a flow rate of 0.5 mL/min. The acquisition method in positive-ion mode with Dual Agilent Jet Stream electrospray voltage used a capillary temperature of 250 °C, a fragmentor of 175 V, a capillary voltage of 3000 V. Mass deconvolution was performed using Agilent MassHunter Qualitative Analysis B.06.00 software with BioConfirm Workflow.

Tandem mass spectrometry analysis
The samples were precipitated and resolved with 8 M urea, and then digested for 16 h at 25 °C by chymotrypsin at an enzyme-to-substrate ratio of 1:50 (wt/wt). The digested peptides were desalted and loaded onto a homemade 30 cm-long pulled-tip analytical column (ReproSil-Pur C18 AQ 1.9 μm particle size, Dr. Maisch GmbH, 75 μm ID× 360 μm OD) connected to an Easy-nLC1200 UHPLC (Thermo Scientific) for mass spectrometry analysis. The elution gradient and mobile phase constitution used for peptide separation were as follows: 0-1 min, 5%-8% B; 1-114 min, 8-35% B; 115-116 min, 35-50% B; 116-120min, 60-100% B (mobile phase A: 0.1% Formic Acid in Water and mobile phase B: 0.1% formic acid in 80% Acetonitrile) at a flow rate of 300 nL /min. Peptides eluted from the LC column were directly electro-sprayed into the mass spectrometer with the application of a distal 1.8-kV spray voltage. Survey full-scan MS spectra (from m/z 300-1500) were acquired in the Orbitrap analyzer (Eclipse) with resolution r =120,000 at m/z 400. The dynamic exclusion time was set at 30 seconds. One acquisition cycle includes one full-scan MS spectrum followed by top MS/MS events in the cycle time setting at 3 s, sequentially generated on the most intense ions selected from the full MS spectrum at a 30% normalized collision energy. The acquired MS/MS data were analyzed UniProtKB E. coli database (database released on Nov. 11, 2016) containing nsp5 using Protein Discoverer 2.4. In order to accurately estimate peptide probabilities and false discovery rates (FDR), we used a decoy database containing the reversed sequences of all the proteins appended to the target database.
FDR was set at 0.01. Mass tolerance for precursor ions was set at 20 ppm. Chymotrypsin was defined as cleavage enzyme and the maximal number of missed cleavage sites was set at 4. Protein N-terminus acetylation, methionine oxidation and compounds covalent bindings were set as variable modifications. The modified peptides were manually checked and labeled.

The synthesis of PF-07321332
The starting material (amine salt 1) were prepared according to the literature procedure (Venkatraman et al., 2006).